Ion channels composed of pore‐forming and auxiliary subunits control physiological functions in virtually all cell types. Whether the multi‐subunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and rapid modulation to control physiological cellular functions is unclear. Arterial myocytes express large‐conductance Ca2+‐activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BK α subunits are primarily (~95%) plasma membrane‐localized, whereas only a small fraction (~10%) of total β1 subunits are located at the cell surface in human and rat arterial myocytes. Immuno‐FRET microscopy demonstrated that intracellular β1 subunits are stored within Rab11A‐postive recycling endosomes. Nitric oxide (NO), acting via protein kinase G, stimulated rapid (1 min) anterograde trafficking of β1 subunit‐containing endosomes. These β1 subunits associated with surface‐resident BKα proteins, elevating Ca2+‐sensitivity and inducing activation. Data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid surface trafficking of β1 subunits controls functional BK channel activity in arterial myocytes and vascular contractility. Grant Funding Source: Supported by NIH‐HL67061

A subset of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) show complex karyotype (CK), and these cases include a relatively high proportion of cases of therapy-related myeloid neoplasms and TP53 mutations. We aimed to evaluate the clinicopathologic features of outcome of 299 AML and MDS patients with CK. Mutations were present in 287 patients (96%) and the most common mutation detected was in TP53 gene (83%). A higher frequency of TP53 mutations was present in therapy-related cases (p=0.008) with a trend for worse overall survival (OS) in therapy-related patients as compared with de novo (p=0.08) and within the therapy-related group, the presence of TP53 mutation strongly predicted for worse outcome (p=0.0017). However, there was no difference in survival between CK patients based on categorization of AML versus MDS, (p=0.96) or presence of absence of circulating blasts ≥1% (p=0.52). TP53 mutated patients presented with older age (p=0.06) and lower hemoglobin (p=0.004) and marrow blast (p=0.02) compared to those with CK lacking TP53 mutation. Multivariable analysis identified presence of multi-hit TP53 mutation as strongest predictor of worse outcome, while neither a diagnosis of AML versus MDS nor therapy-relatedness independently influenced OS. Our findings suggest that among patients with MDS and AML, the presence of TP53 mutation (in particular multi-hit TP53 mutation) in the context of CK identifies a homogeneously aggressive disease, irrespective of the blast count at presentation or therapy-relatedness. The current classification of these cases into different disease categories artificially separates a single biologic disease entity.

Rationale Voltage-dependent L-type (CaV1.2) Ca2+ channels are a heteromeric complex formed from pore forming α1 and auxiliary α2δ and β subunits. CaV1.2 channels are the principal Ca2+ influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte CaV1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of α2δ subunits. Objective Investigate the functional significance of α2δ subunits in myocytes of resistance-size (100–200 μm diameter) cerebral arteries. Methods and Results α2δ-1 was the only α2δ isoform expressed in cerebral artery myocytes. Pregabalin, an α2δ-1/-2 ligand, and an α2δ-1 antibody, inhibited CaV1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95 % of CaV1.2 α1 and α2δ-1 subunits are present in the arterial myocyte plasma membrane. α2δ-1 knockdown using shRNA reduced plasma membrane-localized CaV1.2 α1 subunits, caused a corresponding elevation in cytosolic CaV1.2 α1 subunits, decreased intracellular Ca2+ concentration, inhibited pressure-induced vasoconstriction (“myogenic tone”), and attenuated pregabalin-induced vasodilation. Prolonged (24 hour) pregabalin exposure did not alter total α2δ-1 or CaV1.2 α1 proteins, but decreased plasma membrane expression of each subunit, which reduced myogenic tone. Conclusions α2δ-1 is essential for plasma membrane expression of arterial myocyte CaV1.2 α1 subunits. α2δ-1 targeting can block CaV1.2 channels directly and inhibit surface expression of CaV1.2 α1 subunits, leading to vasodilation. These data identify α2δ-1 as a novel molecular target in arterial myocytes, manipulation of which regulates contractility.

Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with the symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca2+ concentration, and depletion of intracellular Ca2+ by BAPTA or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of Ask1 or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased Tyr-phosphorylation of occludin, ZO-1, E-cadherin and β-catenin. SP600125 abrogated DSS-induced Tyr-phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto phosphorylation of c-Src. This study demonstrates that Ca2+-Ask1-MKK7-JNK2-cSrc signaling cascade mediates DSS-induced tight junction disruption and barrier dysfunction.

A ctivation of plasma membrane phospholipase (PL)Ccoupled receptors by vasoconstrictor agonists leads to phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis and the generation of inositol-1,4,5-trisphosphate (IP 3 ) and diacylglycerol. 1 In vascular myocytes, diacylglycerol (DAG) activates protein kinase (PK)C, leading to the phosphorylation of a wide variety of proteins, including ion channels. 2 IP 3 binds to sarcoplasmic reticulum (SR) IP 3 receptors (IP 3 Rs), resulting in SR Ca 2ϩ release, an elevation in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), and vasoconstriction. 3 Recent evidence also indicates that IP 3 -induced vasoconstriction can occur independently of SR Ca 2ϩ release and via the activation of type 1 IP 3 receptors (IP 3 R1) and type 3 canonical transient receptor potential (TRPC) channels. 4,5 However, the functional signaling mechanisms by which IP 3 Rs and TRPC channels communicate in arterial myocytes are unclear.The mammalian TRP channel superfamily is encoded by at least 28 different genes that are subdivided into 7 families. 6 These families encode ion channels with diverse ion selectivity, modes of regulation, and physiological functions. 6 Vascular myocytes express at least 4 TRP families, including TRPC, TRPM, TRPV, and TRPP. [7][8][9][10] These channels regulate arterial myocyte membrane potential, [Ca 2ϩ ] i , contractility, and proliferation, and are implicated in the etiology of vascular diseases. 4,8 -12 Given the diversity of vascular myocyte TRP channels, it has become important to identify signaling pathways that Original received July 15, 2009; resubmission received January 8, 2010; revision received March 22, 2010; accepted March 29, 2010. From the Department of Physiology, University of Tennessee Health Science Center, Memphis. Correspondence to Jonathan H. Jaggar, Department of Physiology, University of Tennessee Health Science Center, 894 Union Ave, Nash Building, Memphis, TN 38139. E-mail jjaggar@uthsc.edu © 2010 American Heart Association, Inc. Thus, TRPC3 and TRPC6 channels perform distinct physiological functions, but signaling pathways that mediate this differential regulation are unclear.Here, we studied mechanisms by which IP 3 R1, the principal molecular and functional arterial myocyte IP 3 R isoform, 5 stimulates TRPC currents in resistance-size cerebral arteries. Data suggest that IP 3 R1 is in close spatial proximity to, and associates with, TRPC3, but not TRPC6 or TRPM4 channels. Endothelin (ET)-1, a PLC-coupled receptor agonist, and IP 3 alter the interaction between the IP 3 R N terminus and the TRPC3 channel C terminus, leading to channel activation and vasoconstriction. Data indicate that IP 3 R1 selectively couples to TRPC3 channels because of the close spatial proximity of these proteins and that this mechanism is essential for mediating ET-1 and IP 3 -induced vasoconstriction. Methods Tissue PreparationAnimal protocols used were reviewed and approved by the Animal Care and Use Committee at the University of Tennessee Health Science ...